1. Field of the Invention
The present invention relates to a reagent for analyzing leukocytes and a method for classifying leukocytes. In particular, it relates to a reagent for analyzing and counting leukocytes and a method for classifying and counting leukocytes in clinical testing fields.
2. Description of the Related Art
It is important to classify and count leukocytes using the blood of a patient to diagnose various diseases during a clinical examination.
Among leukocytes, basophils contained in a normal sample make up only 0.5% to 2.0% of all leukocytes. When classifying and counting leukocytes, leukocytes other than basophils are lysed or the nuclei thereof are made naked, leaving basophils specifically remaining. Then, an electric impedance signal or a scattered light signal of a cell is measured and a difference in the intensity of the signals is detected, thereby distinguishing basophils from other subclasses of leukocytes.
Japanese Laid-open Patent Publication Nos. Hei 3 (1991)-20667, Hei 4 (1992)-230854 and Sho 61 (1986)-88896 disclose such a method.
Japanese Laid-open Patent Publication No. Hei 3 (1991)-20667 discloses a method of measuring an electric impedance signal of a cell (the DC method) by using an aqueous solution having a pH value of 3.0 to 4.0 which comprises a nonionic surfactant having an additional molar number of polyoxyethylene of 12 to 30 and a cationic surfactant.
Japanese Laid-open Patent Publication No. Hei 4 (1992)-230854 discloses that an electric impedance signal of a cell is measured by using an aqueous solution having a pH value of 2.5 to 3.2 which comprises a polyoxyethylene-type nonionic surfactant, SDS, phthalic acid-hydrochloric acid and an anti-oxidant.
Japanese Laid-open Patent Publication No. Sho 61 (1986)-88896 discloses that basophils can be counted by measuring light scattered at a low angle scattered and light scattered at a high angle scattered from a cell using an aqueous solution having a pH value of 1.8 to 2.3 which comprises a dilute acid and a water soluble surfactant.
According to Japanese Laid-open Patent Publication No. Hei 3 (1991)-20667, basophils can be classified and counted by making the nuclei of leukocytes other than basophils and immature granulocytes naked, and the appearance of immature granulocytes can be detected by the DC method. However, as the data of immature granulocytes and basophils are over-lapped, immature granulocytes and basophils can not be classified and counted separately, and other subclasses of leukocytes also can not be classified and counted. In comparison with FIGS. 12 and 13, even when the reagent disclosed in this reference is applied to a flow cytometer to measure the light scattered from a cell at a low angle and at a high angle, leukocytes can not be classified into subclasses such as immature granulocytes, basophils and the like.
Japanese Laid-open Patent Publication No. Hei 4 (1992)-230854 merely discloses a method to classify and count basophils and fails to suggest the counting of other subclasses of leukocytes as well as the classification and counting of myeloblasts and immature granulocytes.
Japanese Laid-open Patent Publication No. Sho 61 (1986)-88896 enables one to detect the appearance of blast cells, immature granulocytes and left shift by classifying and counting basophils, mononuclear cells (lymphocytes and monocytes) and PMN (neutrophils and eosinophils) by making the nuclei of leukocytes other than basophils naked, and by comparing the morphological features of nuclei (the intensity of the high angle scattered light) and the peroxidase channel. According to this method, immature granulocytes are detected by the difference in the number of mononuclear cells counted in the peroxidase channel because immature granulocytes are included in a fraction of mononuclear cells. Therefore, immature granulocytes can not be detected by using a single channel.